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Endocrinology Vol. 139, No. 3 1075-1081
Copyright © 1998 by The Endocrine Society

ARTICLES

Telomerase Activity in Normal Adult Brown Norway Rat Seminal Vesicle: Regional Distribution and Age-Dependent Changes1

Partha P. Banerjee, Subhadra Banerjee, Barry R. Zirkin and Terry R. Brown

Division of Reproductive Biology, Department of Population Dynamics, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Partha P. Banerjee, Division of Reproductive Biology, Department of Population Dynamics, Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: titli{at}welchlink.welch.jhu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Telomerase activity is essential for protection of cells against the telomere erosion that occurs with each round of cell replication, and thus appears to play a role in the indefinite replication potential of some, but not all, eukaryotic cells. In this regard, some tissues contain stem cells that have a long proliferative life-span and are capable of regenerating or renewing the somatic epithelial cell population within the tissue. Because the adult seminal vesicle exhibits the ability to regenerate during androgen-replacement after castration, we hypothesized that a pool of cells with regenerating potential is present in the adult seminal vesicle, which expresses telomerase activity. In this study, we used a highly sensitive PCR-based telomerase assay [the telomeric repeat amplification protocol (TRAP) assay] to detect telomerase activity in rat seminal vesicle. Our results show that telomerase activity is, indeed, present in the normal adult rat seminal vesicle, but that, in the presence of seminal vesicle fluid, telomerase activity cannot be detected. In fact, seminal vesicle fluid was found to contain some factor(s) that is inhibitory for the TRAP assay. In addition, we found that telomerase activity in the seminal vesicle changes with age and is regionally distributed within the distal, intermediate, and proximal segments of the duct. These results suggest that as is the case for the rat prostate, a population of telomerase-positive cells is present within the adult rat seminal vesicle, and thereby, this organ retains throughout life the potential to regenerate in response to androgen replacement following castration-induced apoptotic cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TELOMERASE activity is essential for protection of cells against the telomere erosion that occurs with each round of cell replication (1, 2, 3, 4), and thus, when present, maintains a cell’s potential for replication without telomere length being affected (5, 6, 7, 8, 9). Telomerase activity has been detected in germ line, tumor tissues, and established cultured cell lines (8, 9, 10, 11, 12, 13, 14, 15, 16), but has not been detected in most normal human somatic cells (8, 11, 17). These observations suggest that telomerase activity may play a role in the indefinite replication potential of some, but not all, eukaryotic cells.

Some tissues contain stem cells that have a long proliferative life-span and are capable of regenerating or renewing the somatic epithelial cell population within the tissue (8, 11, 12, 15, 18, 19, 20, 21, 22, 23, 24, 25). The self-renewal potential of the male accessory sex organs, such as the prostate and seminal vesicles, is well established in the rat. Although the stem cells present in these male accessory sex organs have not been definitively identified, it has been postulated for many years that differentiated epithelial cells arise from stem cells in these organs (26, 27, 28). Recently, we demonstrated the presence of telomerase activity in the prostate of intact adult rats (29), an organ that is able to regenerate in response to androgen. Because the seminal vesicle also has the capability to regenerate in response to androgen, it would be expected that telomerase-positive self-renewing epithelial cells also would be present in this organ throughout life. Surprisingly, however, telomerase activity has not been detected with the PCR-based telomerase assay [the telomeric repeat amplification protocol (TRAP) assay] in the seminal vesicle of intact adult rats, although, after castration, telomerase activity was detected in this tissue (30).

The seminal vesicle of intact rats contains substantial amounts of fluid. It occurred to us that this fluid might, in some way, interfere with the ability to recognize or detect telomerase activity in whole tissue homogenates, particularly because the stem cell population is assumed to be a relatively small proportion of cells within most tissues with a prominent epithelial component (31, 32). To test this hypothesis, we used a highly sensitive PCR-based telomerase assay (the TRAP assay) to detect telomerase activity in the adult rat seminal vesicle before and after its fluid was extruded.

In this study, we demonstrate that telomerase activity is indeed present in cells of the adult rat seminal vesicle, but that seminal vesicle fluid contains some factor(s) that is inhibitory for the TRAP assay. In addition, we also show for the first time that telomerase activity is age dependent and varies regionally within the ducts of the seminal vesicle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Viral antibody-free male Brown Norway rats of 4 and 24 months of age were obtained from Charles River Breeding Lab. (Wilmington, MA) under special arrangement with the National Institute on Aging (Bethesda, MD). The rats were kept in an air-conditioned room with lights on between 0700 h and 1900 h, housed in microisolator cages, and fed autoclaved standard Purina lab chow (Purina Mills, Inc., Richmond, IN) and water ad libitum. Animal protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Hygiene and Public Health.

Dissection of seminal vesicle
The urogenital complex was dissected from the abdominal cavity of each animal and immersed in ice-cold wash buffer (10 mM HEPES, pH 7.5, containing 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride). The tissue was further rinsed and transferred to a petri dish containing fresh, ice-cold wash buffer. Using a dissection microscope, the seminal vesicles were separated from the prostate, blotted onto filter paper, weighed, and snap frozen under liquid nitrogen. In some cases, seminal vesicle fluid was removed by longitudinal incision of the seminal vesicle and thorough washing in buffer. Seminal vesicle fluid was also collected in microfuge tubes and snap frozen in liquid nitrogen. Some seminal vesicles were microdissected into the distal (the outgrowth from the main duct), intermediate (the main duct), and proximal (duct closest to the urethra) segments before freezing.

Preparation of tissue and seminal fluid extracts
Extracts from tissue and seminal vesicle fluid were prepared as previously described (29). Frozen tissue or frozen seminal vesicle fluid was thawed and immediately homogenized in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, containing 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and incubated on ice for 30 min. Tissue lysates and seminal vesicle fluid extracts were clarified by centrifugation at 14,000 x g for 20 min at 4 C, and the supernatants were flash frozen in liquid nitrogen. An aliquot of supernatant was used for the determination of protein content (33) using the Bio-Rad protein assay reagent (Bio-Rad Labs., Hercules, CA).

TRAP assay
To determine the levels of telomerase activity in seminal vesicle, TRAP assays were performed as described by Kim et al. (11) and Piatyszek et al. (34), with slight modification (29). Detection of telomerase activity in the tissue extracts was performed as a two-step process: first, telomerase-mediated extension of an oligonucleotide (TS: 5'-AAT CCG TCG AGC AGA GTT-3'); and second, PCR amplification of the resultant product with the forward (TS) and reverse (CX: 5'-CCC TTA CCC TTA CCC TTA CCC TAA-3') primers. An aliquot of tissue extract equivalent to 5 µg protein was added to a 40-µl reaction solution containing 50 µM deoxynucleotide triphosphates (PCR nucleotide mix; Perkin-Elmer, Norwalk, CT), 2 U Taq DNA polymerase (GIBCO-BRL, Gaithersburg, MD), 1 µg T4 gene 32 protein (Boehringer Mannheim, Indianapolis, IN), 0.1 µg TS primer, and 2 µCi 32P-labeled deoxycytidine triphosphate (Amersham, Arlington Heights, IL). The reaction was incubated for 30 min at 23 C and then heated to 90 C for 3 min. After telomerase synthesized TTAGGG repeats onto the TS primer, 0.1 µg CX primer was added into each tube, and the reaction products were amplified by 27 cycles using a DNA thermal cycler (Perkin-Elmer). Each cycle was run under the following conditions: 94 C for 30 sec, 50 C for 30 sec, and 72 C for 1.5 min. DNA products were separated by electrophoresis on 10% polyacrylamide gels in 0.5x TBE (45 mM Tris-borate, pH 8.3, 1 mM EDTA), pH 8.3, at 300 V for 2.5 h. The gels were dried and exposed to Hyperfilm MP (Amersham, Little Chalfont, England) with an intensifying screen for 16–18 h at -70 C. The ladder of radioactive DNA fragments represents the PCR-amplification of the telomeric repeat sequences synthesized by the telomerase enzyme. Because telomerase activity was shown to be sensitive to RNase treatment, indicating that one of the components of the complex was RNA (3, 35), we pretreated lung and seminal vesicle tissue extracts with RNase A as a negative control.

Statistical analyses
Because lung had the highest telomerase activity compared with any other tissues we examined (29), relative telomerase activity in each experiment was determined as the percentage of radioactivity for a given tissue sample compared with that of lung (=100%). Radioactivity was quantitated by cutting the area corresponding to the entire ladder for telomerase activity from the dried polyacrylamide gel and counting it by scintillation spectrophotometry. Data are expressed as the mean ± the SE of the mean. Statistical differences between two groups (young vs. old seminal vesicles or seminal vesicles with fluid vs. without fluid) were determined by Student’s t test (P < 0.05). More than two groups (e.g. various segments of the seminal vesicle) were analyzed by one-way ANOVA followed by the Scheffé’s F test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As an accessory sex organ, the seminal vesicle produces large amounts of fluid, which is necessary for the ejaculation of sperm during copulation. Table 1Go shows the wet weights for the left and right lobes of the seminal vesicle from six different adult rats, as well as the weights of the right lobe after the fluid was expelled. It is clear from these data that approximately 50% of the wet tissue weight is due to the fluid in this organ.


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  		Table 1. Wet weights of seminal vesicles with or without fluid

 
Other investigators previously demonstrated that telomerase activity was absent from the seminal vesicle of adult intact rats, but was present in the seminal vesicle of castrated rats (30). Because 50% of the total tissue weight of the seminal vesicle in adult intact rats is due to the fluid volume, it seemed possible that telomerase activity within the tissue was greatly diluted by the seminal vesicle fluid. To examine this possibility, we assayed telomerase activity in the seminal vesicles from intact adult rats, either with or without the fluid. In the tissue extracts from all six adult rats, we detected telomerase activity when the fluid was removed before homogenization of the gland, but not when the fluid remained in the tissue (Fig. 1AGo). Lung and heart tissue extracts were used as positive and negative controls, respectively. Quantitatively, young adult seminal vesicle contained substantial telomerase activity equivalent to 40% of the lung activity (Fig. 1BGo).



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  		Figure 1. Telomerase activity in extracts from seminal vesicles with or without fluid. A, Autoradiograph of radiolabeled DNA on polyacrylamide gels. Equal amounts of protein (5 µg) were used in each sample. B, Quantitative analysis of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Lung and heart tissue extracts were used as positive and negative controls. Mean ± SEM for seminal vesicle samples from six different rats.

 
To verify that the activity being measured was genuine telomerase activity, we preincubated the tissue extracts from lung (positive control) and the fluid-free seminal vesicle with RNase A. In both tissue preparations, preincubation with RNase A destroyed telomerase activity completely (Fig. 2Go).



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  		Figure 2. Authencity of telomerase activity in extracts from lung and seminal vesicle of Brown Norway rat. An autoradiograph of radiolabeled DNA on a polyacrylamide gel showing equal amounts of protein (5 µg) from lung (positive control) and seminal vesicle (experimental tissue) without or with pretreatment by RNase A. S. vesicle, Seminal vesicle.

 
Thus, it is clear from our present study that telomerase activity is present in the rat seminal vesicle. Furthermore, the level of telomerase activity present within the seminal vesicle in the absence of fluid suggested that the inability to detect telomerase activity in the presence of fluid was not simply due to dilution. This raised the possibility that seminal vesicle fluid may contain some factor(s) that inhibits the TRAP assay reaction. To explore this possibility, we performed a mixing experiment in which we added increasing amounts (1–10 µg) of seminal vesicle fluid protein to a fixed amount (5 µg) of lung protein extract (positive control). We observed a dose-dependent decrease of telomerase activity in the lung extract, such that 1 µg of seminal vesicle fluid protein caused an 85% reduction in telomerase activity (Fig. 3Go, A and B). To determine whether the addition of protein per se interferes with the telomerase assay, an equal amount of BSA was added in complementary assays. The addition of BSA did not change telomerase activity compared with the control. This result confirms that seminal vesicle fluid contains an inhibitory factor(s) for telomerase activity as measured by the TRAP method.



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  		Figure 3. Inhibitory effects of seminal vesicle fluid on telomerase activity in tissue extracts from lung. Telomerase activity was assayed in lung tissue extract (5 µg protein) in absence or presence of increasing amounts of BSA (1–10 µg protein) and seminal vesicle fluid (1–10 µg protein). A, A representative autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based on equal amounts of protein. B, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean of two separate experiments using seminal vesicle fluid from two different rats. Lung Ext., Lung extract; SV Fluid, Seminal vesicle fluid.

 
To determine whether age-dependent changes occur in telomerase activity, seminal vesicles from five young (4 months old) and five old (24 months old) rats were assayed independently (Fig. 4Go). Telomerase activity was present in samples from both young and old rats (Fig. 4AGo). Based on quantitative analysis, there was a more than 2-fold increase in telomerase activity as a function of age (Fig. 4BGo). Compared with the activity present in the positive control tissue extract from lung (100%), telomerase activity in the young and old seminal vesicles was 48% and 130%, respectively. Telomerase activity was undetectable in the negative control tissue extracts from heart.



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  		Figure 4. Age-dependent increase of telomerase activity in seminal vesicle from Brown Norway rats. Telomerase activity was measured in extracts of fluid extruded from seminal vesicles of young (4 months) and old (24 months) rats. A, Autoradiograph of radiolabeled DNA on a polyacrylamide gel from assays based on equal amounts of protein (5 µg). B, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands after electrophoresis. Mean ± SEM for tissue samples from five different rats.

 
We previously demonstrated that regional differences occur in telomerase activity of the ventral, dorsal, lateral, and anterior prostatic lobes (29). Therefore, we determined whether telomerase activity differed between the distal, intermediate, and proximal segments of the seminal vesicles in young and old rats. Figure 5AGo shows a whole-mount view of the adult seminal vesicle. Figure 5BGo is a diagram showing the locations of the distal, intermediate, and proximal segments within a seminal vesicle. The distal tip segments are small outgrowths from the main duct, and therefore multiple small fragments were combined for determination of telomerase activity. The intermediate segment is the main body and largest portion of the seminal vesicle. The proximal segment is the portion of the seminal vesicle that extends from the urethra to the first ductal branch point. In both young (Fig. 5Go, C and D) and old (Fig. 5Go, E and F) rats, telomerase activity was highest in the distal segment, with less in the intermediate segment, and very little in the proximal segment. Based on quantitative analyses of radioactivity (Fig. 5Go, D and F), there was a significantly greater level (1.5-fold) of telomerase activity in the distal segment of old than of young rats, but significantly less (3-fold) activity in the proximal segment. Telomerase activity was similar in the intermediate segments at both ages.



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  		Figure 5. Regional differences of telomerase activity in fluid extruded from seminal vesicles of young and old Brown Norway rats. A, Whole-mount view of microdissected seminal vesicle from an adult rat. B, Diagrammatic view of seminal vesicle, showing distal (dotted areas), intermediate (empty area), and proximal (starred area) segments. C, Autoradiograph of radiolabeled DNA on a polyacrylamide gel from seminal vesicles of young rats. D, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands from seminal vesicles of young rats. E, Autoradiograph of radiolabeled DNA on a polyacrylamide gel from seminal vesicles of old rats. F, Quantitative analyses of DNA fragments assessed by scintillation counting of radiolabeled DNA bands from seminal vesicles of old rats. Telomerase assays were based on equal amounts of protein (5 µg). Lung and heart tissue extracts were used as positive and negative controls, respectively. Mean ± SEM for tissue samples from three different rats. Dist, Distal segment; Inter, Intermediate segment; Prox, Proximal segment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The activation of telomerase activity in adult human somatic cells has been demonstrated to occur in tumors and is associated with cellular immortalization (6, 8, 9, 10, 11, 14). However, many tissues with a major epithelial cell component, such as the prostate, seminal vesicle, breast, ovary, lung, and intestine contain cells with the potential for self-renewal. Such tissues would be expected to possess a cell population that is immortal, or in any case, has a longer proliferative life-span than other somatic cells (19, 31, 32). Stem cells have self-renewing potential, retain proliferative capability, and are telomerase positive. However, except for skin and intestine, most of the adult somatic tissues in humans are telomerase negative. In contrast, in adult rats, telomerase activity has been detected in many tissues, including adrenal, liver, lung, spleen, and small intestine (29). We recently demonstrated telomerase activity in the ventral, dorsal, lateral, and anterior prostatic lobes of adult rats (29). We do not know whether all of the organs in rats that we (29) and others (30) have shown to be telomerase positive also have self-renewing potential. The presence of telomerase activity plays a role in the potential for cell replication when it is needed for tissue regeneration or immortalization. This has been proposed in the case of mouse cells, which express a high level of telomerase activity, and coincidentally, immortalize in culture easily as compared with human cells (11, 36, 37).

In the present study, we demonstrate the presence of telomerase activity in normal adult rat seminal vesicle, but only if seminal vesicle fluid is extruded before homogenization of the tissue. We propose, therefore, that a previous observation that telomerrase activity was absent in the seminal vesicles of intact adult rats but present in the seminal vesicles of castrated rats (30), probably is related to the presence of fluid in the seminal vesicles of intact rats which inhibited the TRAP assay. Castration also increases the relative proportion of telomerase-positive cells (possibly stem cells) within the tissue, making telomerase activity easier to detect. Indeed, we show that seminal vesicle fluid contains a factor(s) that is inhibitory for the TRAP assay; using lung tissue extract as the positive control, we found a dose-dependent decrease in telomerase activity on the addition of seminal vesicle fluid. In fact, telomerase activity in the lung samples was reduced by 85% when only 1 µg protein equivalent of seminal vesicle fluid was added to the assay mixture. A similar result was also obtained when seminal vesicle fluid was added to the fluid-free seminal vesicle tissue extract (data not shown). These results clearly suggest that seminal fluid contains a potent inhibitory factor(s) for the TRAP assay. At this point, we do not know the characteristics of this factor(s), and thus further investigation will be required to determine the mechanism for this inhibition. We are particularly interested in determining whether this factor(s) is specific for the telomerase enzyme complex because as yet the factors that regulate telomerase activity are unknown. Moreover, a telomerase-specific inhibitor could have significance as an anticancer agent. There are several possibilities by which seminal vesicle fluid could inhibit the TRAP assay: 1) specific inhibition of the telomerase enzyme complex; 2) inhibition of Taq polymerase activity in the TRAP assay; 3) nonspecific proteolytic degradation of the telomerase enzyme; or 4) RNase activity that degrades the RNA component of the telomerase enzyme complex.

Results from the present study also demonstrated that telomerase activity in the rat seminal vesicle increases with age. The age-dependent increase in telomerase activity is not an isolated event that occurs only in the seminal vesicle. For example, in adrenal, lung, and the dorsal and anterior prostatic lobes, we also observed an age-dependent increase in telomerase activity (our unpublished results). We do not know whether these organs are more susceptible to the development of cancer with age, but there is evidence in the literature that a high frequency of spontaneous immortalization occurs in rodent cells in vitro, and a high frequency of cancer occurs in mice in vivo on a per cell per year basis (38). It will be interesting to investigate whether immortalization of cells in culture correlates with higher telomerase activity in these aged rodent organs.

Because we observed regional differences in telomerase activity within the ducts of the various prostatic lobes from young adult rats (29), we also examined the regional distribution of telomerase activity in the seminal vesicles from young and old rats. Telomerase activity was highest in the distal segment > intermediate segment > proximal segment in both young and old rats. This regional distribution of telomerase activity differed from the distribution we observed in prostatic lobes where the highest activity was detected in the proximal segment (29). It is important to note that although the TRAP assay can detect very low levels of telomerase activity in cells or tissue extracts, this assay has certain limitations for quantitation of telomerase activity, especially from tissue extracts that may contain inhibitory factors for the TRAP assay. To eliminate this possibility, we extruded seminal vesicle fluid before homogenization of the tissue in each case. Such inhibitory factors could be present in many other tissues, and therefore appropriate measure must be taken to eliminate such inhibitors before the determination of telomerase activity. A recent modification of the TRAP assay (39) is to coamplify a control DNA fragment of known size by competitive PCR using the identical primers as for amplification of telomeric repeats. Whereas this modification detects the presence of inhibitors based on decreased amplification of the control DNA, the determination of telomerase activity by the competitive PCR reaction provides only semiquantitative results. At this point, it is not possible to determine the specific cell types that express telomerase activity in the seminal vesicle of young and old rats, and we do not as yet know whether regional functional differences exist. With the development of rat telomerase specific complementary DNA/complementary RNA probes for in situ hybridization, or antibodies for immunocytochemical localization, the question of telomerase localization will be resolved in the future.

In summary, our data clearly demonstrate that telomerase activity is present in the seminal vesicles of intact adult rats, and shows age-dependent and region-specific differences. The absence of telomerase activity in the seminal vesicle as reported previously (30) was due to inhibition of the TRAP assay by a factor(s) in seminal vesicle fluid. The expression of telomerase activity in androgen-dependent tissues of the male reproductive tract, such as the prostate and seminal vesicle, and in other organs as observed in this and previous studies (29), suggests that many tissues in rodents have reserve regenerating cells and thereby the potential for self-renewal throughout life.


   Footnotes
 
1 This work was supported by NIH Grant AG-08321. Back

Received September 15, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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